This invention relates to materials and methods for detecting and modulating the activity of RNA. The invention generally relates to the field of "antisense" compounds, compounds which are capable of specific hybridization with a nucleotide sequence of an RNA. In accordance with preferred embodiments, this invention is directed to the design, synthesis and application of oligonucleotides and to methods for achieving therapeutic treatment of disease, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins and cleaving RNA in site specific fashions.
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as "antisense" agents. The oligonucleotides or oligonucleotide analogs complementary to a specific, target, messenger RNA, mRNA sequence are used. A number of workers have reported such attempts. Pertinent reviews include C. A. Stein & J. S. Cohen, Cancer Research, vol. 48, pp. 2659-2668 (1988); J. Walder, Genes & Development, vol. 2, pp. 502-504 (1988); C. J. Marcus-Sekura, Anal. Biochemistry, vol. 172, 289-295 (1988); G. Zon, Journal of Protein Chemistry, vol. 6, pp-131-145 (1987); G. Zon, Pharmaceutical Research, vol. 5, pp. 539-549 (1988); A. R. Van der Krol, J. N. Mol, & A. R. Stuitje, BioTechniques, vol. 6, pp. 958-973 (1988) and D. S. Loose-Mitchell, TIPS, vol. 9, pp. 45-47 (1988). Each of the foregoing provide background concerning general antisense theory and prior techniques.
Thus, antisense methodology has been directed to the complementary hybridization of relatively short oligonucleotides to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs which are designed to bind in a specific fashion to--which are specifically hybridizable with--a specific mRNA by hybridization. Such analogs are intended to inhibit the activity of the selected mRNA--to interfere with translation reactions by which proteins coded by the mRNA are produced--by any of a number of mechanisms. The inhibition of the formation of the specific proteins which are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits.
A number of chemical modifications have been introduced into antisense oligonucleotides to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA, and to improve their pharmacokinetic properties. At present, however, no generalized antisense oligonucleotide therapeutic or diagnostic scheme has been found. The most serious deficiency of prior efforts has been the complete lack of a termination event once appropriate hybridization takes place or the presence of only a termination event that is so inefficient that a useful potency cannot be achieved due to the inability of oligonucleotides to be taken into cells at effective concentrations. The activity of the antisense oligonucleotides presently available has not been sufficient for effective therapeutic, research reagent, or diagnostic use in any practical sense. Accordingly, there has been and continues to be a long-felt need for oligonucleotides and oligonucleotide analogs which are capable of effective therapeutic and diagnostic antisense use.
This long-felt need has not been satisfied by prior work in the field of antisense oligonucleotide therapy and diagnostics. Others have failed to provide materials which are, at once, therapeutically or diagnostically effective at reasonable rates of application.
Initially, only two mechanisms or terminating events have been thought to be operating in the antisense approach to therapeutics. These are the hybridization arrest mechanism and the cleavage of hybridized RNA by the cellular enzyme, ribonuclease H (RNase H). It is likely that additional "natural" events may be involved in the disruption of targeted RNA, however.
These naturally occurring events are discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989). The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides; P. S. Miller & P.O.P. Ts'O, Anti-Cancer Drug Design, 2:117-128 (1987), and .alpha.-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second "natural" type of terminating event is the activation of RNase H by the heteroduplex formed between the DNA type oligonucleotide and the targeted RNA with subsequent cleavage of target RNA by the enzyme. The oligonucleotide or oligonucleotide analog, which must be of the deoxyribo type, hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of antisense agents which are thought to operate by this type of antisense terminating event. R. Y. Walder and J. A. Walder, in Proceedings of the National Academy of Sciences of the U.S.A., Vol. 85, pp.5011-5015 (1988) and C. A. Stein, C. Subasinghe, K. Shenozuka, and J. Cohen, in Nucleic Acids Research, Vol 16, pp.3209-3221 (1988) describe the role the RNase H plays in the antisense approach.
To increase the potency via the "natural" termination events the most often used oligonucleotide modification is modification at the phosphorus atoms. One oligonucleotide analog that has been developed in an effort to secure hybridization arrests is a methyl phosphonate oligonucleotide. Such analogs of oligonucleotides, analogs in the sense that the ordinary structure of the oligonucleotide has been modified into one or more methylphosphonate-substituted structures, have been extensively reported on. A number of authors including K. L. Agarwal & F. Riftina, Nucleic Acids Research, vol. 6, pp. 3009-3024 (1979); P. S. Miller, J. Yano, E. Yano, C. Carroll, C. Jayaraman, K. & P.O.P. Ts'o, Biochemistry, vol. 18, pp. 5134-5143 (1979); K. Jayaraman, K. McParland & P.O.P. Ts'o, Proceedings of the National Academy of Sciences of the U.S.A., vol. 78, pp. 1537-1541 (1981); P. S. Miller, K. B. McParland, K. Jayaraman and P.O.P. Ts'o, Biochemistry, vol. 20, pp. 1874-1880 (1981); P. S. Miller, K. N. Fang, N. S. Kondo and P.O.P. Ts'o, Journal of the American Chemical Society, vol. 93, pp. 6657-6665 (1971); C. H. Agris, K. R. Blake, P. S. Miller, M. P. Reddy, and P.O.P. Ts'o, Biochemistry, vol. 25, 6268-6275 (1986); C. C. Smith, L. Aurelian, M. P. Reddy, P. S. Miller & P.O.P. Ts'o, Proceedings of the National Academy of Sciences of the U.S.A., vol. 83, pp. 2787-2791 (1986); and S. W. Ruby and J. Abelson, Science, vol. 242, pp. 1028-1035 (1988) have reported on such oligonucleotide modifications. In such modifications, a non-bonding phosphoryl oxygen of the phosphorodiester linking moiety is replaced or the nucleotide elements together are replaced, either in total or in part, by methyl groups. This modification gives the molecule a greater resistance to nucleases. Inhibition of gene expression has been demonstrated with methylphosphonate oligonucleotides targeted to several mRNA's as reported, for example, by D. M. Tidd, T. Hawley, H. M. Warenius & I. Gibson, AntiCancer Drug Design, vol. 3, pp. 117-127, (1988). Methyl phosphonate modified oligonucleotides do not activate RNase H on hybridization to RNA and only operate in a strictly hybridization arrest mode. Oligonucleotides from this modification class typically possess inferior binding to targeted RNA, likely due to an R/S stereoisomer at each phosphorus atom, as well as increased steric bulk about the phosphate linkage.
Other workers have made other types of modifications to the phosphorus atom of the phosphate backbone of oligonucleotides in attempts to increase the efficiency of oligonucleotide therapy. The most prominent example is the use of phosphorothioate oligonucleotides. These have included HCPF Roelen, E. DeVroom, G. A. Van der Marel & J. H. VanBoom, Nucleic Acid Research, vol. 16, pp. 7633-7645 (1988) who employed methyl phosphorothionates. S. Agarwal, J. Goodchild, M. P. Civeira, A. H. Thornton, P. S. Sarin & P. C. Zamecnik, in Proceedings of the National Academy of Sciences of the U.S., vol. 85, pp. 7079-7083 (1988); M. Matsukura, K. Shinozuka, G. Zon, H. Mitsuya, M. Reitz, J. S. Cohen & S. Broder, in Proceedings of the National Academy of Sciences of the U.S., vol. 84, pp. 7706-7710 (1987); and C. J. Marcus-Sekura, A. M. Woerner, K. Shinozuka, G. Zon & G. V. Quinnan, in Nucleic Acid Research, vol. 15, pp. 5749-5763 (1987) employed phosphorothioates. See also Biosis/CA Selects abstract 110:88603e, reflecting U.S. Ser. No. 30,075 filed Sep. 1, 1987 which relates to phosporothioate modified oligonucleotides. The phosphorothioate modified oligonucleotides are thought to terminate RNA by activation of RNase H upon hybridization to RNA although hybridization arrest of RNA function may play some part in their activity.
Phosphorodithioates for such use have been disclosed by W. K. D. Brill, J. Y. Yang, Y. X. Ma, & M. H. Caruthers, Journal of the American Chemical Society, vol. 111, pp. 2321-2322 (1989). The phosphorothioate and phosphorodithioate type modifications possess a non-antisense mode of action in that they bind to and inhibit protein function. Protein interactions of oligonucleotides of this type unfortunately undermines the concept of the antisense approach which originally attracted researchers to this novel area. In addition, phosphoroamidates have been disclosed for such uses by A. Jager, M. J. Levy and S. M. Hecht in Biochemistry, vol. 27, pp. 7237-7246 (1988); and R. L. Letsinger, S. A. Bach & J. S. Eadie, in Nucleic Acids Research, vol. 14 pp. 3487-3499 (1986).
In contemplating the application of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, all applications of oligonucleotides as diagnostic, research reagents, and potential therapeutic agents require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides toward nuclease degradation.
A serious deficiency of oligonucleotides for these purposes, particularly antisense therapeutics, is the enzymatic degradation of the administered oligonucleotide by a variety of ubiquitous nucleolytic enzymes, intracellularly and extracellularly located, hereinafter referred to as "nucleases". It is unlikely that unmodified, "wild type", oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases. Modification of oligonucleotides to render them resistant to nucleases is therefore currently a primary focus of antisense research.
Modifications of oligonucleotides to enhance nuclease resistance have heretofore exclusively taken place on the sugar-phosphate backbone, particularly on the phosphorus atom. Phosphorothioates, methyl phosphonates, phosphorimidates, and phosphorotriesters (phosphate methylated DNA) have been reported to have various levels of resistance to nucleases. However, while the ability of an antisense oligonucleotide to bind to specific DNA or RNA with fidelity is fundamental to antisense methodology, modified phosphorous oligonucleotides, while providing various degrees of nuclease resistance, suffer from inferior hybridization properties.
Phosphorus atom modifications such as methyl phosphonates, phosphorothioates, and phosphoramidates, as described above, confer chirality at the phosphorus atom. Due to this prochiral nature of the phosphorous atom, modifications on the internal phosphorus atoms of modified phosphorous oligonucleotides result in Rp and Sp stereoisomers. Since a practical synthesis of stereoregular oligonucleotides (all Rp or Sp phosphate linkages) is unknown, oligonucleotides with modified phosphorus atoms have n.sup.2 isomers with n equal to the number of the phosphorus atoms in the oligonucleotide so modified. Furthermore, modifications on the phosphorus atom have unnatural bulk about the phosphorodiester linkage which interferes with the conformation of the sugar-phosphate backbone and consequently, effects the stability of the duplex. The effects of phosphorus atom modifications cause inferior hybridization to the targeted nucleic acids relative to the unmodified oligonucleotide hybridizing to the same target.
The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T.sub.m), a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coil (unhybridized) forms are present. T.sub.m is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T.sub.m. The higher the T.sub.m, the greater the strength of the binding of the strands. Non-Watson-Crick base pairing has a strong destabilizing effect on the T.sub.m. Consequently, absolute fidelity of base pairing is necessary to have optimal binding of an antisense oligonucleotide to its targeted RNA.
Considerable reduction in the hybridization properties of methyl phosphonates and phosphorothioates has been reported by Cohen. Methyl phosphonates have a further disadvantage in that the duplex formed with RNA does not activate degradation by RNase H as an terminating event, but instead acts by hybridization arrest which can be reversed due to a helical melting activity located on the ribosome. Phosphorothioates are highly resistant to most nucleases. However, phosphorothioates typically exhibit non-antisense modes of action, particularly the inhibition of various enzyme functions due to nonspecific binding. Enzyme inhibition by sequence-specific oligonucleotides undermines the very basis of antisense chemotherapy.
While known modifications to oligonucleotides have been shown to have some effect on improving their inhibition of translation, and while such materials have shown some inhibitory activity towards the production of proteins coded by the mRNA, activities which are sufficient for diagnostic or therapeutic use have not been demonstrated.
Oligonucleotides modified to exhibit resistance to nucleases, to activate the RNase H terminating event, and to hybridize with appropriate strength and fidelity to its targeted RNA (or DNA) are still greatly desired for antisense oligonucleotide purposes.
M. Ikehara et al., European Journal of Biochemistry 139:447-450(1984) report the synthesis of a mixed octamer containing one 2'-deoxy-2'-fluoroguanosine residue or one 2'-deoxy-2'-fluoroadenine residue. W. Guschlbauer and K. Jankowski, Nucleic Acids Res. Vol. 8, p. 1421 (1980) have shown that the contribution of the N form (3'-endo, 2'-exo) increases with the electronegativeness of the 2'-substituent. Thus, 2'-deoxy-2'-fluorouridine contains 85% of the C3'-endo conformer. M. Ikehara et al., Tetrahedron Letters Vol. 42, p. 4073 (1979) have shown that a linear relationship between the electronegativeness of 2'-substituents and the % N conformation (3'-endo-2'-exo) of a series of 2'-deoxyadenosines. M. Ikehara et al., Nucleic Acids Research Vol. 5, p. 1877 (1978) have chemically transformed 2'-deoxy-2'-fluoro-adenosine to its 5'-diphosphate. This was subsequently enzymatically polymerized to provide poly(2'-deoxy-2'-fluoroadenylic acid).
Furthermore, evidence was presented which indicates that 2'-substituted 2'-deoxyadenosines polynucleotides resemble double stranded RNA rather than DNA. M. Ikehara et al., Nucleic Acids Res. Vol. 5, p. 3315 (1978) show that a 2'-fluorine substituent in poly A, poly I, and poly C duplexed to their U, C, or I complement are significantly more stable than the ribo or deoxy poly duplexes as determined by standard melting assays. M. Ikehara et al., Nucleic Acids Res. 4:4249 (1978) show that a 2'-chloro or bromo substituients in poly(2'-deoxyadenylic acid) provides nuclease resistance. F. Eckstein et al., Biochemistry Vol. 11, p. 4336 (1972) show that poly(2'-chloro-2'-deoxyuridylic acid) and poly(2'-chloro-2'-deoxycytidylic acid) are resistant to various nucleases. H. Inoue et al., Nucleic Acids Research Vol. 15, p.6131 (1987) describe the synthesis of mixed oligonucleotide sequences containing 2'-OMe at every nucleotide unit. The mixed 2'-OMe substituted sequences hybridized to their ribooligonucleotide complement (RNA) as strongly as the ribo-ribo duplex (RNA-RNA) which is significantly stronger than the same sequence ribo- deoxyribo heteroduplex (T.sub.m s, 49.0 and 50.1 versus 33.0 degrees for nonamers). S. Shibahara et al., Nucleic Acids Research Vol. 17, p. 239 (1987) describe the synthesis of mixed oligonucleotides sequences containing 2'-OMe at every nucleotide unit. The mixed 2'-OMe substituted sequences were designed to inhibit HIV replication.
It is thought that the composite of the hydroxyl group's steric effect, its hydrogen bonding capabilities, and its electronegativeness versus the properties of the hydrogen atom is responsible for the gross structural difference between RNA and DNA. Thermal melting studies indicate that the order of duplex stability (hybridization) of 2'-methoxy oligonucleotides is in the order of RNA-RNA, RNA-DNA, DNA-DNA. The 2'-deoxy-2'-halo, azido, amino, methoxy homopolymers of several natural occurring nucleosides have been prepared by polymerase processes. The required 2'-modified nucleosides monomers have not been incorporated into oligonucleotides via nucleic acids synthesizer machines. Thus, mixed sequence (sequence-specific) oligonucleotides containing 2'-modifications at each sugar are not known except for 2'-deoxy-2'-methoxy analogs.
Other synthetic terminating events, as compared to hybridization arrest and RNase H cleavage, have been studied in an attempt to increase the potency of oligonucleotides for use in antisense diagnostics and therapeutics. Thus, an area of research has developed in which a second domain to the oligonucleotide, generally referred to as a pendant group, has been introduced.
The pendant group is not involved with the specific Watson-Crick hybridization of the oligonucleotide analog with the mRNA but is carried along by the oligonucleotide analog to serve as a reactive functionality. The pendant group is intended to interact with the mRNA in some manner more effectively to inhibit translation of the mRNA into protein. Such pendant groups have also been attached to molecules targeted to either single or double stranded DNA.
The type of pendant group known as an intercalating agent has been disclosed by Cazenave, N. Loreau, N. T. Thuong, J. J. Toulme and C. Helene in Nucleic Acid Research, vol. 15, pp. 4717-4736 (1987) and J. F. Constant, P. Laugaa, B. P. Roques & J. Lhomme, in Biochemistry, vol. 27, pp. 3997-4003 (1988). The disclosed purpose of such intercalating agents is to add binding stability to the hybrid formed between the oligonucleotide analog and the target nucleic acid by binding to the duplex formed between them.
It has also been disclosed to provide a pendant group to oligonucleotide analogs which is capable of cross-linking. Thus, a pendant agent such as psoralin has been disclosed by A. T. Yeung, B. K. Jones and C. T. Chu in Biochemistry, vol. 27, pp. 2304-3210 (1988). It is believed that after hybridization of the oligonucleotide analog to the target mRNA, the psoralin is photoactivated to cross-link with the mRNA forming a covalent bond between the oligonucleotide analog and the mRNA thereby permanently inactivating the mRNA molecule and precluding the further formation of protein coded by that particular portion of RNA.
It has also been proposed to employ an alkylating agent as a pendant group for oligonucleotide analogs for use in antisense approaches to diagnostics and therapeutics as disclosed by R. B. Meyer in the Journal of American Chemical Society, Vol. 111, pp 8517-8519 (1989) and D. G. Knorre an V. V. Vlassov, Progress in Nucleic Acid Research and Molecular Biology, Vol.32, pp.291-320 (1985).
The object of employing alkylating agents and pendant groups in oligonucleotide analogs in antisense approaches is to cause the alkylating agent to react irreversibly with the target mRNA. Such irreversible binding between the antisense oligonucleotide and the mRNA is generally covalent and leads to permanent inactivation of the mRNA with a concomitant halt in protein production from the portion of mRNA thus inactivated.
A further strategy which has been proposed is to use chemical reagents which, under selected conditions, can generate a radical species for reaction with the target nucleic acid to cause cleavage or otherwise to inactivate it. Proposed pendant groups of this category include coordination complexes containing a metal ion with associated ligands. A metal ion can change oxidation state to generate reactive oxygen-containing radical ions or other radical species. P. L. Doan, L. Perrouault, M. Chassignol, N. T. Thuong, & C. Helene, in Nucleic Acids Research, Vol. 15, pp. 8643-8659 (1987) have disclosed iron/EDTA and iron/porphrin species for this purpose. Copper/phenanthroline complexes have been disclosed by D. S. Sigman, in Accounts of Chemical Research, Vol. 19, pp. 180-186 (1986). G. B. Dreyer and P. B. Dervan, in Proceedings of the National Academy of Sciences, U.S.A., Vol. 82, pp.968-972 (1985) have investigated the EDTA/Fe moiety to cleave nucleic acids.
Prior approaches using cross-linking agents, alkylating agents, and radical generating species as pendant groups on oligonucleotides for antisense diagnostics and therapeutics have several significant shortcomings. The sites of attachment of the pendant groups to oligonucleotides play an important, yet imperfectly known, part in the effectiveness of oligonucleotides for therapeutics and diagnostics. Prior workers have described most pendant groups as being attached to a phosphorus atom which, as noted above, affords oligonucleotides with inferior hybridization properties. Prior attempts have been relatively insensitive, that is the reactive pendant groups have not been effectively delivered to sites on the messenger RNA molecules for alkylation or cleavage in an effective proportion. Moreover, even if the reactivity of such materials were perfect, i.e. if each reactive functionality were to actually react with a messenger RNA molecule, the effect would be no better than stoichiometric. That is, only one mRNA molecule would be inactivated for each molecule of oligonucleotide. It is also likely that the non-specific interactions of the modified oligonucleotides with molecules other then the target RNA, for example with other molecules that may be alkylated or which may react with radical species, as well as self-destruction, not only diminishes the diagnostic or therapeutic effect of the antisense treatment but also leads to undesired toxic reactions in the cell or in vitro. This is especially acute with the radical species which are believed to be able to diffuse beyond the locus of the specific hybridization to cause undesired damage to non-target materials, other cellular molecules, and cellular metabolites. This perceived lack of specificity and stoichiometric limit to the efficacy of such prior alkylating agent and radical generating-types of antisense oligonucleotides is a significant drawback to their employment.
Accordingly, there remains a great need for antisense oligonucleotide formulations which are capable of improved specificity and effectiveness both in binding and in mRNA modulation or inactivation without the imposition of undesirable side effects.